Mitochondria-rich Research Articles

Phenotypic Changes in Mitochondrion-Rich Cells and Responses of Na+/K+-ATPase in Gills of Tilapia Exposed to Deionized Water

The aim of this study was to illustrate the phenotypic modification of mitochondrion-rich (MR) cells and Na(+)/K(+)-ATPase (NKA) responses, including relative protein abundance, specific activity, and immunolocalization in gills of euryhaline tilapia exposed to deionized water (DW) for one week. The plasma osmolality was not significantly different between tilapia of the local fresh water (LFW) group and DW group. Remodeling of MR cells occurred in DW-exposed fish. After transfer to DW for one week, the relative percentage of subtype-I (wavy-convex) MR cells with apical size ranging from 3 to 9 microm increased and eventually became the dominant MR cell subtype. In DW tilapia gills, relative percentages of lamellar NKA immunoreactive (NKIR) cells among total NKIR cells increased to 29% and led to significant increases in the number of NKIR cells. In addition, the relative protein abundance and specific activity of NKA were significantly higher in gills of the DW-exposed fish. Our study concluded that tilapia require the development of subtype-I MR cells, the presence of lamellar NKIR cells, and enhancement of NKA protein abundance and activity in gills to deal with the challenge of an ion-deficient environment.

Expression and water calcium dependence of calcium transporter isoforms in zebrafish gill mitochondrion-rich cells

BackgroundFreshwater fish absorb Ca2+ predominantly from ambient water, and more than 97% of Ca2+ uptake is achieved by active transport through gill mitochondrion-rich (MR) cells. In the current model for Ca2+ uptake in gill MR cells, Ca2+ passively enters the cytosol via the epithelium Ca2+ channel (ECaC), and then is extruded into the plasma through the basolateral Na+/Ca2+ exchanger (NCX) and plasma membrane Ca2+-ATPase (PMCA). However, no convincing molecular or cellular evidence has been available to support the role of specific PMCA and/or NCX isoforms in this model. Zebrafish (Danio rerio) is a good model for analyzing isoforms of a gene because of the plentiful genomic databases and expression sequence tag (EST) data.ResultsUsing a strategy of BLAST from the zebrafish genome database (Sanger Institute), 6 isoforms of PMCAs (PMCA1a, PMCA1b, PMCA2, PMCA3a, PMCA3b, and PMCA4) and 7 isoforms of NCXs (NCX1a, NCX1b, NCX2a, NCX2b, NCX3, NCX4a, and NCX4b) were identified. In the reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, 5 PMCAs and 2 NCXs were ubiquitously expressed in various tissues including gills. Triple fluorescence in situ hybridization and immunocytochemistry showed the colocalization of zecac, zpmca2, and zncx1b mRNAs in a portion of gill MR cells (using Na+-K+-ATPase as the marker), implying a subset of ionocytes specifically responsible for the transepithelial Ca2+ uptake in zebrafish gills. The gene expressions in gills of high- or low-Ca2+-acclimated zebrafish by quantitative real-time PCR analysis showed that zecac was the only gene regulated in response to environmental Ca2+ levels, while zpmcas and zncxs remained steady.ConclusionThe present study provides molecular evidence for the specific isoforms of Ca2+ transporters, zECaC, zPMCA2, and zNCX1b, supporting the current Ca2+ uptake model, in which ECaC may play a role as the major regulatory target for this mechanism during environmental challenge.

Regulation of glycogen metabolism in gills and liver of the euryhaline tilapia (Oreochromis mossambicus) during acclimation to seawater

Glucose, which plays a central role in providing energy for metabolism, is primarily stored as glycogen. The synthesis and degradation of glycogen are mainly initialized by glycogen synthase (GS) and glycogen phosphorylase (GP), respectively. The present study aimed to examine the glycogen metabolism in fish liver and gills during acute exposure to seawater. In tilapia (Oreochromis mossambicus) gill, GP, GS and glycogen were immunocytochemically colocalized in a specific group of glycogen-rich (GR) cells, which are adjacent to the gill's main ionocytes, mitochondrion-rich (MR) cells. Na+/K+-ATPase activity in the gills, protein expression and/or activity of GP and GS and the glycogen content of the gills and liver were examined in tilapia after their acute transfer from freshwater (FW) to 25 per thousand seawater (SW). Gill Na+/K+-ATPase activity rapidly increased immediately after SW transfer. Glycogen content in both the gills and liver were significantly depleted after SW transfer, but the depletion occurred earlier in gills than in the liver. Gill GP activity and protein expression were upregulated 1-3 h post-transfer and eventually recovered to the normal level as determined in the control group. At the same time, GS protein expression was downregulated. Similar changes in liver GP and GS protein expression were also observed but they occurred later at 6-12 h post-transfer. In conclusion, GR cells are initially stimulated to provide prompt energy for neighboring MR cells that trigger ion-secretion mechanisms. Several hours later, the liver begins to degrade its glycogen stores for the subsequent energy supply.

Juvenile Tilapia (Oreochromis mossambicus) Strive to Maintain Physiological Functions After Waterborne Copper Exposure

Juvenile tilapia were acutely exposed to 0.2 and 2 mg/L Cu(2+) for up to 144 h. The Na(+)-K(+)-ATPase (NKA)-specific activity in the gills of tilapia exposed to 0.2 mg/L Cu(2+) significantly decreased over 48-72 h and was restored to the control level after 96 h, but was again depressed during 120-144 h. The whole-body Cl(-) levels significantly decreased after 48 h, but recovered shortly afterwards and continued to do so until 144 h with 0.2 mg/L Cu exposure. During 48-72 h, the numbers of the wavy-convex type of mitochondria-rich (MR) cells appeared to significantly increase and the cortisol content also significantly increased. Changes in MR cell morphology might be necessary in order to enhance Cl(-) uptake, and this might be related to changes in cortisol levels. Whole-body Na(+) concentrations had significantly decreased by 72 h, but recovered during 96-144 h. Whole-body Cu(2+) concentrations also significantly increased compared to the initial concentration during 72-144 h of Cu exposure. All measured parameters (NKA activity, Na(+) concentration, and MR cell numbers) significantly decreased in fish exposed to 2 mg/L Cu, and no recovery was observed. These data demonstrate that juvenile tilapia strived to maintain physiological functions after exposure to sub-lethal concentrations of Cu.

Interactions between Na+ channels and Na+-HCO3− cotransporters in the freshwater fish gill MR cell: a model for transepithelial Na+ uptake

Isolated mitochondria-rich (MR) cells from the rainbow trout gill epithelium were subjected to intracellular pH (pH(i)) imaging with the pH-sensitive dye BCECF-AM. MR cells were categorized into two distinct functional subtypes based on their ability to recover pH(i) from an NH(4)Cl-induced acidification in the absence of Na(+). An apparent link between resting pH(i) and Na(+)-independent pH(i) recovery was made. We observed a unique pH(i) acidification event that was induced by extracellular Na(+) addition. This further classified the mixed MR cell population into two functional subtypes: the majority of cells (77%) demonstrated the Na(+)-induced pH(i) acidification, whereas the minority (23%) demonstrated an alkalinization of pH(i) under the same circumstances. The focus of this study was placed on the Na(+)-induced acidification and pharmacological analysis via the use of amiloride and phenamil, which revealed that Na(+) uptake was responsible for the intracellular acidification. Further experiments revealed that pH(i) acidification could be abolished when Na(+) was allowed entry into the cell, but the activity of an electrogenic Na(+)-HCO(3)(-) cotransporter (NBC) was inhibited by DIDS. The electrogenic NBC activity was supported by a DIDS-sensitive, Na(+)-induced membrane potential depolarization as observed via imaging of the voltage-sensitive dye bis-oxonol. We also demonstrated NBC immunoreactivity via Western blotting and immunohistochemistry in gill tissue. We propose a model for transepithelial Na(+) uptake occurring via an apical Na(+) channel linked to a basolateral, electrogenic NBC in one subpopulation of MR cells.

Visualization in zebrafish larvae of Na+uptake in mitochondria-rich cells whose differentiation is dependent onfoxi3a

Uptake of Na(+) from the environment is an indispensable strategy for the survival of freshwater fish, as they easily lose Na(+) from the plasma to a diluted environment. Nevertheless, the location of and molecules involved in Na(+) uptake remain poorly understood. In this study, we utilized Sodium Green, a Na(+)-dependent fluorescent reagent, to provide direct evidence that Na(+) absorption takes place in a subset of the mitochondria-rich (MR) cells on the yolk sac surface of zebrafish larvae. Combined with immunohistochemistry, we revealed that the Na(+)-absorbing MR cells were exceptionally rich in vacuolar-type H(+)-ATPase (H(+)-ATPase) but moderately rich in Na(+)-K(+)-ATPase. We also addressed the function of foxi3a, a transcription factor that is specifically expressed in the H(+)-ATPase-rich MR cells. When foxi3a was depleted from zebrafish embryos by antisense morpholino oligonucleotide injection, differentiation of the MR cells was completely blocked and Na(+) influx was severely reduced, indicating that MR cells are the primary sites for Na(+) absorption. Additionally, foxi3a expression is initiated at the gastrula stage in the presumptive ectoderm; thus, we propose that foxi3a is a key gene in the control of MR cell differentiation. We also utilized a set of ion transport inhibitors to assess the molecules involved in the process and discuss the observations.

Cadmium and calcium uptake in isolated mitochondria-rich cell populations from the gills of the freshwater rainbow trout

A novel cell isolation technique was used to characterize cadmium and calcium uptake in distinct populations of gill cells from the adult rainbow trout (Oncorhynchus mykiss). A specific population of mitochondria-rich (MR) cell, termed the PNA+ MR cell (PNA is peanut lectin agglutinin), was found to accumulate over threefold more 109Cd than did PNA- MR cells, pavement cells (PV cells), and mucous cells during a 1-h in vivo exposure at 2.4 microg/l 109Cd. In vitro 109Cd exposures, performed in standard PBS and Cl- -free PBS, at concentrations from 1 to 16 microg/l 109Cd, were also carried out to further characterize Cd2+ uptake kinetics. As observed during in vivo experiments, PNA+ MR cells accumulated significantly more 109Cd than did other cell types when exposures were performed by an in vitro procedure in PBS. Under such conditions, Cd2+ accumulation kinetics in all cell types could be described with Michaelis-Menten relationships, with Km values of approximately 3.0 microg/l Cd (27 nM) for both MR cell subtypes and 8.6 microg/l Cd (77 nM) for PV cells. In similar experiments performed in Cl- -free conditions, a significant reduction in 109Cd accumulation in PNA+ MR cells was seen but not in PNA- MR or in PV cells. In vitro 45Ca fluxes were also performed to determine the cellular localization of Ca2+ transport in these functionally distinct populations of gill cells. 45Ca uptake was most pronounced in PNA+ MR cells, with levels over threefold higher than those found in either PNA(-) MR or in PV cells. Results from the present study suggest that the PNA+ MR cell type is a high-affinity and high-capacity site for apical entry of Cd2+ and Ca2+ in the gill epithelium of rainbow trout.

Cellular composition and ultrastructure of the gill epithelium of larval and adult lampreys: implications for osmoregulation in fresh and seawater.

Lampreys, one of the only two surviving groups of agnathan (jawless) vertebrates, contain several anadromous species that, during their life cycle, thus migrate from fresh to seawater and back to freshwater. Lampreys have independently evolved the same overall osmoregulatory mechanisms as the gnathostomatous (jawed) and distantly related teleost fishes. Lamprey gills thus likewise play a central role in taking up and secreting monovalent ions. However, the ultrastructural characteristics and distribution of their epithelial cell types [ammocoete mitochondria-rich (MR) cell, intercalated MR cell, chloride cell and pavement cell] differ in several respects from those of teleosts. The ultrastructural characteristics of these cells are distinctive and closely resemble those of certain ion-transporting epithelia in other vertebrates, for which the function has been determined. The data on each cell type, together with the stage in the life cycle at which it is found, i.e. whether in fresh or seawater, enable the following proposals to be made regarding the ways in which lampreys use their gill epithelial cells for osmoregulating in hypo- and hypertonic environments. In freshwater, the intercalated MR cell takes up Cl- and secretes H+, thereby facilitating the uptake of Na+ through pavement cells. In seawater, the chloride cell uses a secondarily active transcellular transport of Cl- to provide the driving force for the passive movement of Na+ through leaky paracellular pathways between these cells.

Enzymatic changes in mitochondria-rich cells of Xenopus laevis skin epithelium are induced by ionic acclimation

Morphological, biochemical and histochemical components of mitochondria-rich (MR) cells of skin epithelium of Xenopus laevis (Daudin) were investigated after acclimation in distilled water (DW) and mild solutions (50 mmol/l) of either NaCl or KCl for over 10 days. The animals readily acclimated to NaCl, but approximately 50% of the animals died in the KCl solution. Electrophysiological measurements confirmed the poor transport properties of skin in all conditions. Silver staining and exposure to methylene blue (MB) have shown that two types of MR cells can be distinguished, especially after KCl acclimation. Immunohistochemistry with the use of anti-band 3 polyclonal and anti H +-ATPase monoclonal antibodies demonstrated that band 3 and H +-ATPase enzymes were localized in MR cells in all conditions. H +-ATPase was greatly reduced during NaCl acclimation as verified with SDS gel electrophoresis. Intensity of the immunohistochemical staining differed between the various conditions of acclimation. Histochemical localization of carbonic anhydrase and alkaline phosphatase activities was more intense during NaCl acclimation. Morphological changes were also observed between the various acclimation conditions. The present findings substantiate the existence of at least two forms of MR cells in Xenopus skin epithelium but their functional significance remains to be established.

Vacuolar-type proton pump in the basolateral plasma membrane energizes ion uptake in branchial mitochondria-rich cells of killifish Fundulus heteroclitus, adapted to a low ion environment.

We examined the involvement of mitochondria-rich (MR) cells in ion uptake through gill epithelia in freshwater-adapted killifish Fundulus heteroclitus, by morphological observation of MR cells and molecular identification of the vacuolar-type proton pump (V-ATPase). MR cell morphology was compared in fish acclimated to defined freshwaters with different NaCl concentrations: low (0.1 mmol l(-1))-, mid (1 mmol l(-1))- and high (10 mmol l(-1))-NaCl environments. MR cells, mostly located on the afferent-vascular side of the gill filaments, were larger in low- and mid-NaCl environments than in the high-NaCl environment. Electron-microscopic observation revealed that the apical membrane of well-developed MR cells in low- and mid-NaCl environments was flat or slightly projecting, and equipped with microvilli to expand the surface area exposed to these environments. On the other hand, in the high-NaCl environment, the apical membrane was invaginated to form a pit, and MR cells often formed multicellular complexes with accessory cells, although the NaCl concentration was much lower than that in plasma. We cloned and sequenced a cDNA encoding the A-subunit of killifish V-ATPase. The deduced amino acid sequence showed high identity with V-ATPase A-subunits from other vertebrate species. Light-microscopic immunocytochemistry, using a homologous antibody, revealed V-ATPase-immunoreactivity in Na(+)/K(+)-ATPase-immunoreactive MR cells in low-NaCl freshwater, whereas the immunoreactivity was much weaker in higher NaCl environments. Furthermore, immuno-electron microscopy revealed V-ATPase to be located in the basolateral membrane of MR cells. These findings indicate that MR cells are the site responsible for active ion uptake in freshwater-adapted killifish, and that basolaterally located V-ATPase is involved in the Na(+) and/or Cl(-) absorbing mechanism of MR cells.

Localization and characterization of phenamil-sensitive Na+influx in isolated rainbow trout gill epithelial cells

Percoll density-gradient separation, combined with peanut lectin agglutinin (PNA) binding and magnetic bead separation, was used to separate dispersed fish gill cells into sub-populations. Functional characterization of each of the sub-populations was performed to determine which displayed acid-activated phenamil- and bafilomycin-sensitive Na(+) uptake. Analysis of the mechanism(s) of (22)Na(+) influx was performed in control and acid-activated (addition of 10 mmoll(-1) proprionic acid) cells using a variety of Na(+) transport inhibitors (ouabain, phenamil, HOE-694 and bumetanide) and a V-type ATPase inhibitor (bafilomycin). We found that cells migrating to a 1.03-1.05 g ml(-1) Percoll interface [pavement cells (PVCs)] possessed the lowest rates of Na(+) uptake and that influx was unchanged during either bafilomycin (10 nmoll(-1)) treatment or internal acidification with addition of proprionic acid (10 mmoll(-1)). Mitochondria-rich (MR) cells that migrated to the 1.05-1.09 g ml(-1) interface of the Percoll gradient demonstrated acidification-activated bafilomycin and phenamil-sensitive Na(+) influx. Further separation of the MR fraction into PNA(+) and PNA(-) fractions using magnetic separation demonstrated that only the PNA(-) cells (alpha-MR cells) demonstrated phenamil-and bafilomycin-sensitive acid-activated (22)Na(+) uptake. We confirm the coupling of a V-type H(+)-ATPase with phenamil-sensitive Na(+) uptake activity and conclude that high-density alpha-MR cells function in branchial Na(+) uptake in freshwater fish.

Na(+), Cl(-), Ca(2+) and Zn(2+) transport by fish gills: retrospective review and prospective synthesis.

The secondary active Cl(-) secretion in seawater (SW) teleost fish gills and elasmobranch rectal gland involves basolateral Na(+),K(+)-ATPase and NKCC, apical membrane CFTR anion channels, and a paracellular Na(+)-selective conductance. In freshwater (FW) teleost gill, the mechanism of NaCl uptake is more controversial and involves apical V-type H(+)-ATPase linked to an apical Na(+) channel, apical Cl(-)-HCO-3 exchange and basolateral Na(+),K(+)-ATPase. Ca(2+) uptake (in FW and SW) is via Ca(2+) channels in the apical membrane and Ca(2+)-ATPase in the basolateral membrane. Mainly this transport occurs in mitochondria rich (MR) chloride cells, but there is a role for the pavement cells also. Future research will likely expand in two major directions, molded by methodology: first in physiological genomics of all the transporters, including their expression, trafficking, operation, and regulation at the molecular level, and second in biotelemetry to examine multivariable components in behavioral physiological ecology, thus widening the integration of physiology from the molecular to the environmental levels while deepening understanding at all levels.

Redistribution of immunofluorescence of CFTR anion channel and NKCC cotransporter in chloride cells during adaptation of the killifish Fundulus heteroclitus to sea water.

Cellular distribution of cystic fibrosis transmembrane conductance regulator (CFTR) immunofluorescence was detected by monoclonal antibody directed to the C terminus of killifish CFTR (kfCFTR) in chloride cells of fresh water (FW) adapted fish and animals transferred to sea water (SW) for 24h, 48h and 14+ days. Confocal microscopy allowed localization within mitochondria-rich (MR) cells to be determined as superficial (i.e. in the apical membrane) or deeper within the cytoplasm of the cells. In FW, 90 % of MR cells had diffuse kfCFTR immunofluorescence in the central part of the cytosol, with only 8.1 % having apical kfCFTR, which was 6.6+/-0.54 microm below the microridges of surrounding pavement cells. Curiously, FW but not SW pavement cells also had positive immunofluorescence to kfCFTR. After 24h in SW, a time when kfCFTR expression is elevated, a condensed punctate immunofluorescence appeared among 18.8 % of MR cells, 13.4+/-0.66 microm (mean +/- S.E.M.) below the surface of the cells. By 48h, a majority (76.3 %) of MR cells had punctate kfCFTR distribution and the distance from the surface was less (7.8+/-0.2 microm), a distribution approaching the SW-acclimated condition (i.e. all MR cells showing kfCFTR immunofluorescence, 6.1+/-0.04 microm below the surface). In contrast, NKCC immunofluorescence was condensed and localized in lateral parts of MR cell complexes in FW animals and then redistributed to the whole basal cytoplasm after acclimation to SW. CFTR, the anion channel responsible for Cl(-) secretion in marine teleosts, redistributes in MR cells during SW acclimation by condensation of a diffuse distribution below the apical crypt, followed by translocation and insertion in the apical membrane. NKCC, the cotransporter that translocates Cl(-) across the basolateral membrane, moves from an eccentric cytosolic location in FW to a diffuse basolateral localization in SW chloride cells.

Structure–function relationships in the integument of Salamandra salamandra during ontogenetic development

Morphological, cytological and transport properties of the integument of Salamandra salamandra were investigated during natural ontogenetic development, from birth to adult. Three stages were operationally defined: I, larvae, from birth to metamorphosis; II, metamorphosis (judged externally by the colour change and loss of the gills); and III, post-metamorphosis to adult. Pieces of skin were fixed at various stages for immunocytochemical examinations, and the electrical properties were investigated on parallel pieces. Distinct cellular changes take place in the skin during metamorphosis, and lectin (PNA, WGA and ConA) binding indicates profound changes in glycoprotein composition of cell membranes, following metamorphosis. Band 3 and carbonic anhydrase I (CA I) were confined to mitochondria-rich (MR)-like cells, and were detected only in the larval stage. CA II on the other hand, was detected both in MR-like and in MR cells following metamorphosis. The electrical studies show that the skin becomes more tight (transepithelial resistance increases) upon metamorphosis, followed by manifestation of amiloride-sensitive short-circuit current ( I sc) indicating that functional Na + uptake has been acquired. The skin of metamorphosed adults had no finite transepithelial Cl – conductance, and band 3 was not detected in its MR cells. The functional properties of MR-like and MR cells remain to be established.

Isolation and characterization of mitochondria-rich cell types from the gill of freshwater rainbow trout.

A magnetic cell separation technique (MACS) was developed for isolating and characterizing peanut lectin agglutinin positive (PNA(+)) cells from rainbow trout gills. Percoll density separated mitochondria-rich (MR) cells were serially labeled with PNA-FITC and an anti-FITC antibody covalently coupled to a 50-nm iron particle and then applied to a magnetic column. PNA(+) MR cells were enriched to >95% purity. Transmission electron microscopy analysis of both the PNA(+) and PNA negative (PNA(-)) fraction showed that PNA binds to MR chloride cells while the PNA(-) cell fraction is comprised of MR cells with features characteristic of pavement cells. Western blotting demonstrated that both PNA(+) and PNA(-) fractions had high levels of Na(+)-K(+)-ATPase and Sco1 expression; however, relative expression of H(+)-ATPase in PNA(+) and PNA(-) cells demonstrated that untreated fish had twofold higher H(+)-ATPase levels in PNA(-) cells relative to the PNA(+) cells. Furthermore, hypercapnic acidosis significantly increased the relative H(+)-ATPase expression on PNA(-) cells only, whereas metabolic alkalosis had no significant effect.

Peanut lectin binds to a subpopulation of mitochondria-rich cells in the rainbow trout gill epithelium.

Fluorescently labeled peanut lectin agglutinin (PNA-FITC) was used to identify a subtype of mitochondria-rich (MR) cells in the gills of freshwater rainbow trout. In situ binding of PNA-FITC was visualized by inverted fluorescence microscopy and found to bind to cells on the trailing edge of the filament epithelium as demonstrated by differential interference contrast optics. The amount of PNA-FITC binding on the filament epithelium increased with cortisol pretreatment concomitant with an increased chloride cell fractional area as demonstrated by scanning electron microscopy. Dispersed gill cells were isolated by trypsinization and separated using a discontinuous Percoll density gradient. Cells migrating to the 1.06-1.09 g/ml interface were found to be MR as demonstrated by staining with the vital mitochondrial dye 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide and transmission electron microscopy (TEM). However, only approximately 40% of the MR cells were found to bind PNA-FITC. Cortisol pretreatment increased the relative numbers of MR cells isolated from the dispersed gill cell population, but the relative proportions of PNA binding cells remained unchanged. Ultrastructural analysis of isolated cells in the TEM demonstrated that the MR cell fraction was comprised of a mixed population of chloride cells and pavement cells.

Modification of morphology and function of integument mitochondria-rich cells in tilapia larvae (Oreochromis mossambicus) acclimated to ambient chloride levels.

Similar to those of the gills of adults, three types of mitochondria-rich (MR) cells with different morphologies of apical surfaces (wavy convex, shallow basin, and deep hole) were identified on the integument of freshwater-acclimated tilapia larvae (Oreochromis mossambicus). The object of this study is to test the hypothesis that these subtype cells may represent MR cells equipped with variable efficiencies in Cl(-) uptake. Larvae acclimated to low-Cl(-) =0.001-0.007 mM) water developed higher densities of MR cells than those acclimated to high-Cl(-) =7.3-7.9 mM) water. The percentage of wavy-convex-type cells in total MR cells was higher in low-Cl(-)-acclimated larvae than in high-Cl(-)-acclimated larvae, which displayed only deep-hole type. In addition, Cl(-) influx rates of whole larva measured with (36)Cl(-) showed a coincident correlation with MR cell densities, that is, low-Cl(-) larvae displayed higher Cl(-) influx rates than did high-Cl(-) larva, suggesting that tilapia larvae develop a higher density of MR cells with larger apical surfaces (wavy-convex type) to boost Cl(-) uptake in Cl(-)-deficient water. The distinct types of apical surfaces may represent different phases of MR cells that possess different efficiencies of Cl(-) uptake. Increased apical membrane surface areas of MR cells may provide larvae with rapid regulation of Cl(-) before new MR cells differentiate.

Morphology and function of gill mitochondria-rich cells in fish acclimated to different environments.

The objective of this study is to test the hypothesis that morphologically different mitochondria-rich (MR) cells may be responsible for the uptake of different ions in freshwater-adapted fish. Tilapia (Oreochromis mossambicus) were acclimated to high-Ca, mid-Ca, low-Ca, and low-NaCl artificial freshwater, respectively, for 2 wk. Cell densities of wavy-convex, shallow-basin, and deep-hole types of gill MR cells as well as whole-body Ca(2+), Na(+), and Cl(-) influxes were measured. Low-Ca fish developed more shallow-basin MR cells in the gills and a higher Ca(2+) influx than those acclimated to other media. However, fish acclimated to low-NaCl artificial freshwater predominantly developed wavy-convex cells, and this was accompanied by the highest Na(+) and Cl(-) influxes. Relative abundance of shallow-basin and wavy-convex MR cells appear to be associated with changes in Ca(2+) and Na(+)/Cl(-) influxes, suggesting that shallow-basin and wavy-convex MR cells are mainly responsible for the uptake of Ca(2+) and Na(+)/Cl(-), respectively.

Medaka ( Oryzias latipes) as a model for hypoosmoregulation of euryhaline fishes

We examined the hypoosmoregulatory ability of a model fish, medaka ( Oryzias latipes), in relation to the gill-chloride cells or mitochondrion-rich (MR) cells, and to cortisol. When the medaka were transferred from freshwater (FW) to 30‰ seawater (SW), muscle water content decreased by 8% after 2 h and normalized within 1 week. Size and density of MR cells in the gill filament increased after 1–2 weeks in SW. Immersion of medaka in FW containing cortisol (10 μg/ml) for 1–2 weeks also doubled the number of MR cells, and abolished the decrease in muscle water content after SW transfer. In SW-adapted medaka, prolactin (PRL; 10 μg/g body weight) injection reduced muscle water content. However, when cortisol (50 μg/g) was injected simultaneously with PRL, cortisol abolished the decrease in muscle water. We concluded that cortisol plays an important role in the SW adaptation of the medaka, whereas PRL may be involved in ion uptake. Medaka seems to be a good model fish useful for the study of osmoregulatory mechanisms in general.

Immunolocalization of ion-transport proteins to branchial epithelium mitochondria-rich cells in the mudskipper (Periophthalmodon schlosseri).

The branchial epithelium of the mudskipper Periophthalmodon schlosseri is densely packed with mitochondria-rich (MR) cells. This species of mudskipper is also able to eliminate ammonia against large inward gradients and to tolerate extremely high environmental ammonia concentrations. To test whether these branchial MR cells are the sites of active ammonia elimination, we used an immunological approach to localize ion-transport proteins that have been shown pharmacologically to be involved in the elimination of NH(4)(+) (Na(+)/NH(4)(+) exchanger and Na(+)/NH(4)(+)-ATPase). We also investigated the role of carbonic anhydrase and boundary-layer pH effects in ammonia elimination by using the carbonic anhydrase inhibitor acetazolamide and by buffering the bath water with Hepes, respectively. In the branchial epithelium, Na(+)/H(+) exchangers (both NHE2- and NHE3-like isoforms), a cystic fibrosis transmembrane regulator (CFTR)-like anion channel, a vacuolar-type H(+)-ATPase (V-ATPase) and carbonic anhydrase immunoreactivity are associated with the apical crypt region of MR cells. Associated with the MR cell basolateral membrane and tubular system are the Na(+)/K(+)-ATPase and a Na(+)/K(+)/2Cl(-) cotransporter. A proportion of the ammonia eliminated by P. schlosseri involves carbonic anhydrase activity and is not dependent on boundary-layer pH effects. The apical CFTR-like anion channel may be serving as a HCO(3)(-) channel accounting for the acid-base neutral effects observed with net ammonia efflux inhibition.